Formonetin is an isoflavone derived from Astragalus membranaceus (Fisch.) Bunge, of which a sulfonate version, sodium formonetin-3’-sulfonate (ND308), and has been shown to improve water solubility when administering injections. However, the therapeutic effects elicited by ND308 toward acute lung injury (ALI) remain unclear. In our study, we examined the in vivo effects of ND308 in a lipopolysaccharide (LPS)-induced two-hit rat ALI model. Then, to investigate the anti-ALI mechanisms mediated by ND308, LPS-induced injury in human pulmonary microvascular endothelial cells (HMs) was examined in vitro. Continuous ND308 administration (20 mg/kg for 3 days) via the tail vein elicited protective effects in our rat model via changes in overall pulmonary function Hounsfield units, and gross pulmonary pathological indicator the lung coefficient and lung microscopic pathology scores, respectively. Furthermore, tight junction (TJ) protein expression values for claudin 18.1 changed from 0.42 to 0.91, while FAS, p-PDK1, p-STAT3, and PDK1 distribution in nuclei changed from 2.54 to 1.29, 2.26 to 1.07, 2.67 to 1.19, and 2.31 to 1.14, respectively. In in vitro studies, LPS-induced HMs showed similar results; lower FAS, p-PDK1, p-STAT3, and PDK1 distribution in nuclei and higher claudin 18.1 values. When combined, ND308 appeared to alleviate ALI by strengthening claudin 18.1 expression via FAS/PDK1/STAT3 inhibition.